Method for preparing vaccine for anti-HPV 16 infection by pichia yeast expression system

A technology of human papillomavirus and Pichia pastoris, which is applied in antiviral agents, botany equipment and methods, biochemical equipment and methods, etc., and can solve the problems of undisclosed recombinant L1 protein activity, etc.

Active Publication Date: 2009-07-22
SHANGHAI ZERUN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this document does not disclose the nucleotide sequence of the HPV L1 gene synthesized a

Method used

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  • Method for preparing vaccine for anti-HPV 16 infection by pichia yeast expression system
  • Method for preparing vaccine for anti-HPV 16 infection by pichia yeast expression system
  • Method for preparing vaccine for anti-HPV 16 infection by pichia yeast expression system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Synthesis of HPV 16 L1 Gene

[0039] The nucleotide sequence of the 1.5 kb HPV 16 L1 gene was designed according to the codon bias used by Pichia pastoris. See SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 in the sequence listing.

[0040] The above-mentioned full-length genes were commercially synthesized by Jerry Company respectively, and the HPV 16 L1 gene was sequenced using a DNA sequencer #3730.

Embodiment 2

[0041] Example 2 Construction of HPV 16 L1 Pichia pastoris expression vector

[0042] The two ends of the HPV 16 L1 gene whose sequence was SEQ ID NO.1 obtained in Example 1 were loaded into the pUC18 plasmid (Jierui Company) with EcoRI and KpnI restriction sites, named pUC-16L1, and sent to Shanghai Biotech Gong Biological Engineering Co., Ltd. used DNA sequencer 3730 for DNA sequencing.

[0043] Entrust Shanghai Sangon Bioengineering Co., Ltd. to synthesize the primers required for the amplification of HPV 16 L1. The forward primer includes a BstBI restriction endonuclease site and has a nucleotide sequence of 5'-ACTAATTATTCGAAACGATGTCTTTGTGG-3' (as shown in SEQ ID NO.4). The reverse primer includes KpnI restriction endonuclease sites flanking the stop codon and has the nucleotide sequence 5'-AGCGGTACCCTATTACAACTTTCTCTTCTTTC-3' (shown in SEQ ID NO. 5).

[0044] Polymerase chain reaction (PCR) was carried out with the above primer pairs and pUC-16 L1 as DNA template. The P...

Embodiment 3

[0045] Example 3 Construction and screening of HPV 16 L1 Pichia pastoris expression strain

[0046]In order to improve the integration efficiency of the recombinant plasmid on the yeast chromosome, the pPICZA-16 L1 plasmid was single-digested with Sac I restriction endonuclease to linearize it, and the same digestion was performed on the empty plasmid pPICZA as a negative control. Add absolute ethanol to the enzyme digestion reaction solution to precipitate DNA, dissolve the linearized pPICZA-16 L1 fragment with a small amount of double distilled water, and transform Pichia pastoris X-33 ( available from Invitrogen). The electroporation conditions were: 5 micrograms of DNA fragments, a voltage of 1500 volts, a resistance of 25 ohms, and an electric shock time of 5 milliseconds. Electroporation products were plated on YPDS agar containing 200 μg / ml zeocycin, isolated single colonies of transformed cells, and re-inoculated on 1000 μg / ml and 1500 μg / ml zeocycin resistance plates...

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Abstract

The invention discloses a method for preparing a recombinant human papillomavirus 16-typed L1 protein with a pichia pastoris expression system and comprises the steps of loading an HPV 16 L1 gene in accordance with optimal design into an expression carrier, transforming pichia pastoris and cultivating a transformant, thus obtaining the recombinant human papillomavirus 16-typed L1 protein, which is self-assembled into virus-like particles inside a pichia pastoris body; wherein, nucleotide sequences of the HPV 16 L1 gene in accordance with optimal design are shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3. The HPV 16 L1protein that is prepared by the method of the invention has high expressed quantity. The invention further discloses a method for preparing a papillomavirus 16-typed infection vaccine with better activity by utilizing the virus-like particles of the recombinant HPV 16 L1 protein.

Description

technical field [0001] The invention relates to a method for preparing recombinant human papillomavirus type 16 L1 protein by using a Pichia pastoris expression system and using the L1 protein to prepare a vaccine against human papillomavirus type 16 infection. Background technique [0002] Human papillomavirus (HPV) is a small, non-enveloped, double-stranded circular DNA virus belonging to the Polyomavirinae subfamily of the Papovaviridae family. HPV can be transmitted through close contact between humans, causing common warts and anogenital genital warts and other lesions on the skin of the infected person, and is listed as a sexually transmitted disease. In 1995, the International Cancer Research Center announced the results of a study confirming that HPV has a close causal relationship with cervical cancer. It can be seen that HPV infection has become a pathogen that seriously endangers human health. Therefore, the development of efficient and cheap HPV vaccine is of g...

Claims

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Application Information

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IPC IPC(8): C12N15/37C12N15/81C07K14/025A61K39/12A61P35/00A61P31/20
Inventor 张高峡沈琼袁靖宇张千里魏健熊莹华雷建强吴克
Owner SHANGHAI ZERUN BIOTECHNOLOGY CO LTD
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